Abstract Nucleoside analogs are a significant class of anti-cancer agent. As prodrugs, they terminate the DNA synthesis upon transforming to their active triphosphate metabolites. We have encapsulated a biologically activate nucleotide analog (i.e. gemcitabine triphosphate (GTP)), instead of the nucleoside (i.e. gemcitabine) derivative, into a novel Lipid/Calcium/Phosphate nanoparticle (LCP) platform. The therapeutic efficacy of LCP-formulated GTP was evaluated in a panel of human non-small-cell lung cancer (NSCLC) and human pancreatic cancer models after systemic administrations. GTP-loaded LCPs induced cell death and arrested the cell cycle in the S phase. In vivo efficacy studies showed that intravenously injected GTP-loaded LCPs triggered effective apoptosis of tumor cells, significant reduction of tumor cell proliferation and cell cycle progression, leading to dramatic inhibition of tumor growth, with little in vivo toxicity. Broadly speaking, the current study offers preclinical proof-of-principle that many active nucleotide or phosphorylated nucleoside analogs could be encapsulated in the LCP nanoplatform and delivered systemically for a wide variety of therapeutic applications.